Novel Heptaplex PCR-Based Diagnostics for Enteric Fever Caused by Typhoidal Salmonella Serovars and Its Applicability in Clinical Blood Culture

Enteric fever is caused by typhoidal Salmonella serovars (Typhi, Paratyphi A, Paratyphi B, and Paratyphi C). Owing to the importance of Salmonella serovars in clinics and public hygiene, reliable diagnostics for typhoidal serovars are crucial. This study aimed to develop a novel diagnostic tool for typhoidal Salmonella serovars and evaluate the use of human blood for clinically diagnosing enteric fever. Five genes were selected to produce specific PCR results against typhoidal Salmonella serovars based on the genes of Salmonella Typhi. Heptaplex PCR, including genetic markers of generic Salmonella, Salmonella enterica subsp. enterica, and typhoidal Salmonella serovars, was developed. Typhoidal Salmonella heptaplex PCR using genomic DNAs from 200 Salmonella strains (112 serovars) provided specifically amplified PCR products for each typhoidal Salmonella serovar. These results suggest that heptaplex PCR can sufficiently discriminate between typhoidal and nontyphoidal Salmonella serovars. Heptaplex PCR was applied to Salmonella-spiked blood cultures directly and provided diagnostic results after 12- or 13.5-h blood culture. Additionally, it demonstrated diagnostic performance with colonies recovered from a 6-h blood culture. This study provides a reliable DNA-based tool for diagnosing typhoidal Salmonella serovars that may be useful in clinical microbiology and epidemiology.

While DNA-based PCR detection methods for Salmonella Typhi [13,[16][17][18][19] and Paratyphi A [11,20,21] have been reported, few studies have reported similar methods for Salmonella Paratyphi B and Paratyphi C [22,23].Ultimately, these studies using DNA-based PCR detection methods must be applicable for enteric fever diagnostics in clinical microbiology.Despite the numerous advantages of PCR diagnostics, their efficiency of PCR diagnostics for Salmonella identification must be improved to provide more accurate results and prevent biased diagnostic conclusions at the Salmonella serovar level owing to the limited number of target genes and evaluated Salmonella serovars [5,13,20,21,23].
In our previous studies, PCR-based identification methods for Salmonella Typhimurium and Typhi were developed using specific genetic markers selected from comparisons among Salmonella genome sequences [24][25][26].We inferred that PCR using appropriate genetic markers might sufficiently discriminate between specific Salmonella serovars.The present study aimed to develop reliable PCR diagnostics in a single reaction that could efficiently discriminate between typhoidal and non-typhoidal Salmonella serovars.Moreover, we aimed to employ the developed typhoidal Salmonella heptaplex PCR for efficiently diagnosing enteric fever in clinical microbiology laboratories.We believe that this method will enable rapid and reliable diagnosis of typhoidal Salmonella serovars and contribute to improving human health and public hygiene.

Bacterial Strains
A total of 200 Salmonella strains were used, including 112 serovars of Salmonella subspecies I-VI, as listed in Table 1.Sixteen type strains of Salmonella were obtained from the American Type Culture Collection (ATCC).Other Salmonella strains were obtained from the Federal Institute for Risk Assessment (BFR) of Germany [27], US Food and Drug Administration (FDA, CFSAN/OPDFB) [28], Korea Consumer Protection Board (KCPB) [29], Ministry of Food and Drug Safety (MFDS) of Korea, National Culture Collection for Pathogens (NCCP) of Korea, Food-borne pathogen Omics Research Center (FORC) of Korea, and Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Diseases (APFID) in Korea as listed in Table 1.Salmonella strains were inoculated in tryptic soy broth (TSB) and cultured at 37°C under vigorous shaking conditions.

Genomic DNA Extraction
The cultured media of Salmonella strains were harvested in microtubes.Genomic DNA from Salmonella was extracted using a DNeasy Blood & Tissue kit (Qiagen, Germany) according to the manufacturer's instructions.The concentration of the extracted DNA was measured using a UV spectrophotometer (model UV-1700; Shimadzu, Japan), and genomic DNA at a 1.8 to 2 ratio (A 260 /A 280 ) was used.Genomic DNA from Salmonella strains was diluted in distilled water to 25 ng/μl and stored at 4°C prior to use in PCR.

Genetic Markers for Typhoidal Salmonella Serovars and Primer Design
In our study, 195 genes of Salmonella Typhi CT18 (GenBank Accession No. NC_003198) were found to be highly specific to Salmonella genus and serovar Typhi [26].These genes were subjected to the non-redundant (nr) DNA sequence database of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) using the BLAST program [30] to screen for candidate genes specifically present in Salmonella Typhi and Paratyphi A, B, or C. Primers for screening candidate genes were designed and constructed (Bioneer, Korea).

Single PCR Condition
PCR was performed using primers constructed from genomic DNAs of various Salmonella serovars, as listed in Table 1.Each 25 μl PCR mixture contained 1× EX Taq buffer, 0.4 μmol/l primer, 200 μmol/l concentrations of each dNTP, 0.5 Unit of EX Taq DNA polymerase (TaKaRa, Japan), and 25 ng/μl template DNA.PCR amplification was performed in a thermocycler (Model GeneAtlas G, ASTEC, Japan) with an initial denaturation at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 30 s and a final extension at 72°C for 3 min and 4°C for 5 min.Amplified products were electrophoresed on a 2% agarose gel in 0.5× Tris-Acetate-EDTA buffer, stained with DNA staining reagent (NEOscience, Korea), and photographed under UV-irradiation using a Vilber Gel Doc system (KoreaBIOMICS, Korea).

Internal Amplification Control
To generate an Internal Amplification Control (IAC) for verifying of PCR performance, the partial DNA sequence of the tubulin β-4 chain gene (GenBank Accession No. NM_123801) was amplified from the genomic DNA of Arabidopsis thaliana using primers STM3098_F2_ flank_TB (5-TTT GGC GCA GGC GAT TC-CAA TCC AGG AGA TGT TTA GGC G-3), and STM3098_R2_ flank_TB (5-GCC TCC GCC TCA ATC CG-CCT Typhoidal Salmonella Heptaplex PCR Heptaplex PCR for typhoidal Salmonella serovars was performed using the primer sets listed in Table 2. Heptaplex PCR was designed to amplify eight genes targeting generic Salmonella, S. enterica subspecies enterica (I), Salmonella Typhi, Paratyphi A, Paratyphi B, Paratyphi C, and IAC.Heptaplex PCR was performed with seven primer sets at each concentration (Table 2) and IAC-template plasmid (approximately, 5 × 10 7 copies) using AccuPower Multiplex PCR PreMix (K-2111, BIONEER, Korea) in a 20-μl mixture.The reaction conditions were the same as those mentioned in the previous section for single PCR, except initial denaturation at 94°C for 10 min.A amplified were electrophoresed on a 3.5% agarose gel for 100 V, 70 min.The amplified products were analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) equipped with a DNA 1000 LabChip kit (Agilent Technologies).

Preparation of Salmonella-Spiked Blood Culture Samples
Each culture of Salmonella Typhi and Paratyphi A, B, and C was serially diluted in TSB and then added to human whole blood at a concentration of 5 CFU/ml.For the blood culture, 10 ml of Salmonella-spiked blood was inoculated into Bact/ALERT SA Aerobic medium bottle (40 ml) (bioMérieux, INC., Durham, NC 27712, USA) and then cultured in a shaking incubator at 210 rpm at 37°C for up to 24 h.Human whole blood (SER-WB, Lot#: WB082819E) containing K 2 -EDTA as anticoagulant, which was obtained from a healthy volunteer adult donor who has signed an the Institutional Review Board (IRB) validated donor consent form, was purchased from the Zenbio Inc. (Research Triangle Park, NC 27709, USA).Cultured blood samples were collected at 0, 6, 9, 10.5, 12, 13.5, 15, 16.5, 18, 21, and 24-h time points.One milliliter of the collected blood culture was centrifuged at 16,000 ×g for 10 min, and the supernatant was carefully discarded.A total of 200 μl PrepMan Ultra reagent (Life Technologies, USA) was added, boiled for 15 min at 100°C, centrifuged for 5 min at 16,000 ×g, and finally 2 μl of supernatant was added as template DNA for the heptaplex PCR.Additionally, Salmonella colonies were recovered from the collected blood culture at the 6-h point.Blood culture (100 μl) was spread onto tryptic soy agar plate and cultured for 10 h at 37°C to allow Salmonella colony formation.A portion of Salmonella colony was picked using a sterile pipette tip and suspended in 100 μl PrepMan Ultra reagent or TE buffer (pH 8.0).Each sample was boiled for 15 min at 100 o C, and centrifuged for 5 min at 16,000 ×g.Supernatant (2 μl) was added as template DNA for each typhoidal Salmonella heptaplex PCR.Also, a small portion of recovered Salmonella colony was picked using a sterile pipette tip and directly added into the heptaplex PCR (termed as "PCR on colony").

Selection of Genetic Marker for Constructing of Typhoidal Salmonella Heptaplex PCR
In our previous study on selecting novel genetic markers for Salmonella Typhi, 195 genes of Salmonella Typhi CT18 (GenBank Accession No. NC_003198) were screened via comparative genomics, which are present only in Salmonella genus [26].We determined that some of the 195 genes were highly specific for Salmonella Typhi, Paratyphi A, Paratyphi B, or Paratyphi C. In the present study, candidate genetic markers were selected from 195 genes that were highly specific to Salmonella Paratyphi A, Paratyphi B, or Paratyphi C, based on the BLAST output against the NCBI nr database.Primer sets were designed for the selected 13 candidate genes and were evaluated with various genomic DNAs of Salmonella serovars to finalize the selection of specific genetic markers for identifying each Salmonella Paratyphi A, Paratyphi B, and Paratyphi C. Finally, a typhoidal Salmonella heptaplex PCR was constructed, including the selected genetic markers presented in Table 2.The heptaplex PCR included specific genetic markers for Salmonella genus (STM3098, 423 bp) [24,26], Salmonella subspecies I (STM4057, 137 bp) [24,26], Salmonella Typhi (STY1599, 258 bp) [26], Paratyphi A (STY3279, 193 bp and STY2750, 70 bp), Paratyphi B (STY3670, 165 bp), Paratyphi C (STY4578, 291 bp) and IAC (100 bp).IAC amplification was performed using primer set of STM3098.

Specificity of Typhoidal Salmonella Heptaplex PCR
The developed typhoidal Salmonella heptaplex PCR assay was evaluated using 112 Salmonella serovars (200 strains), as shown in Table 1.The heptaplex PCR results demonstrated its specific diagnostics for Salmonella genus, Salmonella subspecies I, and Salmonella Typhi, Paratyphi A, Paratyphi B, and Paratyphi C, respectively as shown in Fig. 1 (panel A).Additionally, the PCR products were analyzed using capillary electrophoresis (Bioanalyzer), as shown in Fig. 1 (panel B).The specific peak(s) of each typhoidal Salmonella serovar [marked with arrows] demonstrated clean amplification of the expected size.All Salmonella Typhi strains showed amplification of all PCR products owing to the presence of all target genes in Salmonella Typhi.However, the 258 bp PCR product is a critical diagnostic indicator of Salmonella Typhi.Salmonella Paratyphi A strains, including clinical and food isolates, showed both amplifications of 193 and 70 bp, respectively.These two simultaneous amplifications are critical for diagnosing of Salmonella Paratyphi A, because some Salmonella serovars, such as serovars Georgia, Montevideo, Ohio, Muenster, and Kentucky, revealed one positive results between the two genetic markers.Salmonella Paratyphi B and Paratyphi C showed specific amplifications at expected sizes of 165 bp and 291 bp, respectively.As expected, all Salmonella strains showed Salmonella specific amplification at 423 bp and all strains belonging to Salmonella subspecies I showed specific amplification at 137 bp.In this study, amplification of IAC in all reactions removed of false negatives.

Performance of Typhoidal Salmonella Heptaplex PCR with Salmonella-Spiked Blood Culture Sample
The developed typhoidal Salmonella heptaplex PCR was employed for existing blood culture systems, which are used globally for diagnosing various infectious diseases, including enteric fever, in clinical microbiology.The extracted DNA solutions from Salmonella-spiked blood culture samples were evaluated as shown in Fig. 2. Blood culture samples spiked with Salmonella Typhi, Paratyphi A, Paratyphi B, and Paratyphi C showed positive heptaplex PCR results after 12-or 13.5-h of blood culture.Additionally, recovered colonies from Salmonella-spiked blood cultures at the 6-h point (PCR on colony) and extracted DNA from recovered colonies using two boiling methods were evaluated using typhoidal Salmonella heptaplex PCR, as shown in Fig. 3.The two boiling methods provided clean amplification with each typhoidal serovar (Fig. 3 panels A and B), and direct PCR on the colony of each typhoidal serovar recovered from blood culture also showed clean amplification (Fig. 3 panel C).These results demonstrate that the developed typhoidal Salmonella heptaplex PCR could be applied to existing blood culture systems in clinics to obtain detailed serovar information among typhoidal Salmonella serovars.

Discussion
Generally, Salmonella spp., particularly Salmonella subspecies I, are considered pathogens of birds and mammals, including humans, despite some host-specific Salmonella serovars.Therefore, identification of Salmonella at the genus and subspecies levels is important for diagnosing salmonellosis in clinics and public hygiene.The typhoidal Salmonella heptaplex PCR developed in this study included two previously described genetic markers (STM3098 and STM4057 gene) for identifying the genus Salmonella and Salmonella subspecies I, respectively [24,26].These genetic markers could provide critical diagnostic information at Salmonella genus and subspecies levels against other infectious pathogens such as pathogenic E. coli.For diagnosing typhoidal Salmonella serovars, heptaplex PCR contains newly developed genetic markers for Salmonella Paratyphi A (STY3279 and STY2750), Paratyphi B (STY3670), and Paratyphi C (STY4578) and a marker for S. Typhi (STY1599) [26].These genetic markers provide   PCR-based diagnostics can be used to directly identify Salmonella Typhi in blood samples [16,18,[31][32][33].However, in these studies, direct DNA extraction from blood without culture (briefly termed "PCR on blood") did not provide stable PCR amplification and sufficient analysis resolution to confirm positive results on agarose gel electrophoresis, which is not suitable for practical application in clinical microbiology.These difficulties may be because of the low number of Salmonella Typhi present in blood samples (0.5-22 CFU/ml) [34] and the failure to recover the genomic DNA of Salmonella directly from the blood sample.To overcome difficulties in early clinical diagnosis enteric fever from human blood samples, a PCR-based method can be used in the existing diagnostic systems in clinical microbiology, particularly in blood culture-based MALDI-TOF MS system [14].
Interestingly, the addition of ox-bile to the blood culture medium (generally TSB medium) enhances the growth rate of Salmonella, inhibiting the bactericidal activity of blood [35].A single amplification PCR method for Salmonella Typhi was applied to a blood culture sample in ox bile-containing TSB medium, demonstrating clean positive results for a 5-h blood culture sample [34].However, in the present study, the ox-bile-containing blood culture method was not employed because the presence of ox-bile in the blood culture medium only allowed the growth of bile resistant bacteria [13].This method is not preferable in existing blood culture-based MALDI-TOF MS diagnostics, because it is a universal diagnostic tool for enteric fever and other infectious diseases in clinical microbiology.However, our heptaplex PCR revealed positive results for 12-or 13.5-h cultured blood samples, as shown in Fig. 2, which could provide early diagnostics for typhoidal Salmonella serovars.We agree that successful PCR-based diagnostics of typhoidal Salmonella in blood depend on the growth rate and number of Salmonella in blood cultures [34].Additionally, PCR results of the recovered colony from the 6-h blood culture (Fig. 3) could provide complementary diagnostics at Salmonella serovar level along with the MALDI-TOF MS system in clinics.
Therefore, a simple diagnostic tool for enteric fever must be developed [5,8,13].In the present study, a typhoidal Salmonella heptaplex PCR with novel genetic markers was developed and evaluated using various Salmonella serovars, demonstrating its performance and specificity for typhoidal Salmonella serovars.Moreover, the performance of this heptaplex PCR was validated using recovered colonies as well as directly extracted DNA from blood culture samples.The results demonstrated that this typhoidal Salmonella heptaplex PCR provides a novel, reliable DNA-based diagnostic tool for Salmonella typhoidal serovars related to public hygiene, including in the fields of clinical microbiology, food safety, and epidemiology, and could potentially help in early diagnosis of enteric fever when combined with existing blood culture processes in clinics.
Number of strains.b BFR, Federal Institute for Risk Assessment; KCPB, Korea Consumer Protection Board; FDA, US Food and Drug Administration (CFSAN/OPDFB); MFDS (Ministry of Food and Drug Safety); NCCP (National Culture Collection for Pathogens); FORC (Food-borne pathogen Omics Research Center); ABB (Asian Bacterial Bank) of APFID (Asia Pacific Foundation for Infectious Diseases).c +, Positive result; -, negative result.

Table 2 . Primer pairs used for typhoidal Salmonella heptaplex PCR and their expected result with typhoidal Salmonella serovars.
TCC TGA ACA TAG CTG TG-3), to generate a 100-bp amplicon.The resulting amplicon was inserted into pGEM-T Easy Vector (Promega Corporation, USA) to generate an IAC template plasmid for use in the typhoidal Salmonella heptaplex PCR.
a +, Positive result; -, Negative result b IAC, Internal amplification control in heptaplex PCR TTC